Dr. Rupendra Shrestha, MSc, PhD - Hualien Tzu Chi Hospital - Researcher

Dr. Rupendra Shrestha

MSc, PhD

Hualien Tzu Chi Hospital

Researcher

Hualien, Hualien | Taiwan

Main Specialties: Biochemical Genetics, Blood Banking - Transfusion Medicine, Clinical & Laboratory Immunology, Infectious Disease, Medical Genetics, Molecular Genetic Pathology, Ophthalmology

Additional Specialties: Human Genetics and Stem Cells

ORCID logohttps://orcid.org/0000-0001-6804-6070


Top Author

Dr. Rupendra Shrestha, MSc, PhD - Hualien Tzu Chi Hospital - Researcher

Dr. Rupendra Shrestha

MSc, PhD

Introduction

A professionally biology-driven researcher with expertise in laboratory medicine, clinical genetics, and stem cells.

Primary Affiliation: Hualien Tzu Chi Hospital - Hualien, Hualien , Taiwan

Specialties:

Additional Specialties:

Research Interests:


View Dr. Rupendra Shrestha’s Resume / CV

Education

Dec 2019
Tzu Chi University
Ph.D. Medical Sciences
Stem Cells
Jun 2014
Sri Ramachandra University
M.Sc. Human Genetics
Medical Genetics
Nov 2011
Nobel College
B.Sc. MLT
Clinical Laboratory Technologist

Experience

Jun 2015
Research Fellow
Cancer Biology
IBAB
Jul 2014
Clinical Molecular Geneticist
Genetic Diagnostics
Manipal Hospital

Publications

8Publications

178Reads

45Profile Views

12PubMed Central Citations

Effective Differentiation and Biological Characterization of Retinal Pigment Epithelium Derived from Human Induced Pluripotent Stem Cells.

Curr Eye Res 2020 Feb 3:1-13. Epub 2020 Feb 3.

Institute of Medical Sciences, Tzu Chi University, Hualien, Taiwan.

Purpose: Human induced pluripotent stem cells (hiPSC)-derived retinal pigment epithelium (RPE) cells are therapeutic cells that have been shown to be promising in the rescue of lost photoreceptors. In this study, we generated hiPSC from human epidermal keratinocytes and subsequently differentiated them into RPE cells to investigate their ability to influence the retinal functions of the Royal College of Surgeon (RCS) rats. Methods: Keratinocytes were reprogrammed to hiPSC using a non-integrating Sendai reprogramming system. Established hiPSCs were differentiated into RPE cells, and complete characterization was performed. Next, the suspension of hiPSC-RPE cells was transplanted into the subretinal space of 3-week-old RCS rats (n = 12). Posttransplantation evaluations were performed using optical coherence tomography (OCT), electroretinography, and immunohistochemical analysis. Results: The hiPSC colonies were identical to embryonic stem-like cells that revealed the expression of pluripotency markers and retention of the normal genome. These cells exhibited the ability to differentiate into an amalgam of germ layers and produce RPE cells. The differentiated RPE cells exhibited an identical pigmented morphology that expressed RPE-specific markers, such as CRALBP, BESTROPHIN, RPE65, and MERTK. At 8 weeks of longitudinal culture, the RPE cells exhibited maximum pigmentation with in vitro phagocytotic activity. Furthermore, transplantation data showed improved retinal function till week 12 post-transplantation and a significantly higher number of rod/cone ratios in transplanted eyes compared to non-surgery control eyes. Conclusion: hiPSC-derived RPE cells exhibited naïve RPE cell properties and functionality that provided trophic support and the transient rescue of photoreceptor cells.

View Article

Download full-text PDF

Source
http://dx.doi.org/10.1080/02713683.2020.1722180DOI Listing
February 2020
1.672 Impact Factor

Generation of hiPSC line TCIERi001-A from normal human epidermal keratinocytes.

Stem Cell Res 2019 12 15;41:101590. Epub 2019 Oct 15.

Institute of Medical Sciences, Tzu Chi University, Hualien 970, Taiwan; Institute of Eye Research, Hualien Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Hualien 970, Taiwan. Electronic address:

Human induced pluripotent stem cell (hiPSC) line TCIERi001-A was generated from normal human epidermal keratinocytes (NHEK) primary cell line with the nonintegrating system using Sendai reprogramming kit. Sendai particles were used to deliver the defined transcription factors that included three vector preparations, such as polycistronic KLF4-OCT3/4-SOX2, cMYC, and KLF4.

View Article

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.scr.2019.101590DOI Listing
December 2019
1 Read
1 Citation
3.929 Impact Factor

Induced pluripotent stem cells and derivative photoreceptor precursors as therapeutic cells for retinal degenerations

10.4103/tcmj.tcmj_147_19

Tzu Chi Medical Journal


The visual impairment associated with inherited retinal degeneration and age-related degeneration of photoreceptors is causing substantial challenges in finding effective therapies. However, induced pluripotent stem cell (iPSC)-derived therapeutic cells such as photoreceptor and retinal pigment epithelium (RPE) cells provide the ultimate options in the rescue of lost photoreceptors to improve the visual function in end-stage degeneration. Retinal cells derived from iPSC are therapeutic cells that could be promising in the field of cell replacement therapy and regenerative medicine. This review presents an overview of the photoreceptor degeneration, methods of iPSC generation, iPSC in retinal disease modeling, summarizes the photoreceptor differentiation protocols, and challenges remained with photoreceptor cell replacement for the treatment of retinal diseases. Thus, the burden and increased incidence of visual impairment emphasizes the need of novel therapy, where iPSC-derived photoreceptor and RPE cells proved to be promising for curing the retinal dysfunction and act as renovation in approach to improve visual function.

View Article
September 2019
2 Reads

Late Response of Antiretroviral Therapy in an HIV-1-Infected Patient due to Hepatitis B and C Coinfections: The First Case Report in Nepal.

Case Rep Med 2019 11;2019:2070973. Epub 2019 Feb 11.

Sukraraj Tropical & Infectious Disease Hospital, Teku, Kathmandu, Nepal.

Aim: Dual coinfection of HCV and HBV in HIV-1-infected population is a leading cause of morbidity and mortality. Also, they share routes of HIV transmission; however, it might be associated with an independent factor like injecting drug use for HCV and unsafe sex for HBV. This case report suggests that hepatitis virus coinfection may lead to late response of antiretroviral therapy (ART) in HIV-1 patients.

Patients And Methods: A 49-year-old male patient visited for the routine follow-up investigation at the National Public Health Laboratory (NPHL), Teku, Nepal. He was an HIV-1-positive injecting drug user (IDU) co-infected with HCV and HBV. The patient was under ART as per the National HIV Testing and Treatment Guidelines 2017, Nepal. Further, serological and viral load testing was performed for confirmation and monitoring therapy, respectively.

Results: It is the first report that highlights the dual coinfection of HCV and HBV in an HIV-1 patient from Nepal. The follow-up investigation shows improved response to ART with an increase in CD4+ cells. However, detectable viral loads indicated for a late response might be due to effects of coinfections or viral interactions.

Conclusions: Dual coinfection is rare; however, it is more serious with poorly defined epidemiology and evolution in an HIV-1-infected population. Thus, universal screening of HBV or/and HCV coinfection in HIV-1-infected population requires immediate implementation for true prevalence, proper management, and early intervention.

View Article

Download full-text PDF

Source
http://dx.doi.org/10.1155/2019/2070973DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6388324PMC
February 2019
11 Reads
1 Citation

Aberrant hiPSCs-Derived from Human Keratinocytes Differentiates into 3D Retinal Organoids that Acquire Mature Photoreceptors.

Cells 2019 01 9;8(1). Epub 2019 Jan 9.

Institute of Medical Sciences, Tzu Chi University, Hualien 970, Taiwan.

Human induced pluripotent stem cell (hiPSC)-derived three-dimensional retinal organoids are a new platform for studying the organoidogenesis. However, recurrent genomic aberration, acquired during generation of hiPSCs, limit its biomedical application and/or aberrant hiPSCs has not been evaluated for generation of differentiated derivatives, such as organoids and retinal pigment epithelium (RPE). In this study, we efficiently differentiated mosaic hiPSCs into retinal organoids containing mature photoreceptors. The feeder-free hiPSCs were generated from the human epidermal keratinocytes that were rapid in process with improved efficiency over several passages and maintained pluripotency. But, hiPSCs were cytogenetically mosaic with normal and abnormal karyotypes, while copy number variation analysis revealed the loss of chromosome 8q. Despite this abnormality, the stepwise differentiation of hiPSCs to form retinal organoids was autonomous and led to neuronal lamination. Furthermore, the use of a Notch inhibitor, DAPT, at an early timepoint from days 29?42 of culture improved the specification of the retinal neuron and the use of retinoic acid at days 70?120 led to the maturation of photoreceptors. hiPSC-derived retinal organoids acquired all subtypes of photoreceptors, such as , and . Additionally, the advanced maturation of photoreceptors was observed, revealing the development of specific sensory cilia and the formation of the outer-segment disc. This report is the first to show that hiPSCs with abnormal chromosomal content are permissive to the generation of three-dimensional retinal organoids.

View Article

Download full-text PDF

Source
http://www.mdpi.com/2073-4409/8/1/36
Publisher Site
http://dx.doi.org/10.3390/cells8010036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6356277PMC
January 2019
43 Reads
4 Citations
5.656 Impact Factor

Electrospun cellulose acetate phthalate nanofibrous scaffolds fabricated using novel solvent combinations biocompatible for primary chondrocytes and neurons.

Tissue Cell 2016 Dec 29;48(6):634-643. Epub 2016 Jul 29.

Department of Human Genetics, Sri Ramachandra University, Chennai, India. Electronic address:

Electrospun nanofibres have been shown to exhibit extracellular matrix (ECM)-like characteristics required for tissue engineering in terms of porosity, flexibility, fibre organization and strength. This study focuses on developing novel cellulose acetate phthalate (CAP) scaffolds by electrospinning for establishing 3-D chondrocyte and neuronal cultures. Five solvent combinations were employed in fabricating the fibres, namely, acetone/ethanol (9:1), dimethylformamide/tetrahydrofuran/acetone (3:3:4), tetrahydrofuran/acetone (1:1), tetrahydrofuran/ethanol (1:1) and chloroform/methanol (1:1). The electrospun fibres were characterized by scanning electron microscopy (SEM) analysis and confirmed to be within the nanometre range. Based on the morphology of the fibers from SEM results, two solvent combinations such as acetone/ethanol and dimethylformamide/tetrahydrofuran/acetone were selected for stabilization as CAP exhibits a pH dependent solubility. Fourier-Transform Infrared (FTIR) analysis revealed the hydrolysis of CAP which was overcome by EDC [1-ethyl-3-(3-dimethylaminopropyl) carbodiimide] and EDC/NHS (N-hydroxysuccinimide) cross-linking resulting in its stability (pH of 7.2) for three months. MTT [3-(4, 5-dimethylthiazol-2-yl)-1, 5-diphenyltetrazolium bromide] assay performed using L6 myoblast confirmed the biocompatibility of the scaffolds. 3-D primary chondrocyte and neuronal cultures were established on the scaffolds and maintained for a period of 10 days. H&E staining and SEM analysis showed the attachment of the chondrocytes and neurons on CAP scaffolds prepared using dimethylformamide/tetrahydrofuran/acetone and acetone/ethanol respectively.

View Article

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.tice.2016.07.007DOI Listing
December 2016
123 Reads
5 Citations
1.553 Impact Factor

Detection of Latent HIV-1 Infection and Drug Resistant Mutation Testing in Nepal: HIV-1 env V3 DNA Sequence and RT Gene (M184V) Mutation

Nepal Journal of Biotechnology

HIV-1 resistance to antiretroviral therapy (ART) is a crucial issue, despite various effective drugs are available for the treatment. Although the viral RNA is suppressed below the detection limit (<50 copies/ml) with the use of potent antiviral drugs, the mutation can be archived in the cellular reservoir as proviral DNA. The detection of proviral DNA and mutation screening in HIV 1 RNA for genotypic resistance is the sole basis for monitoring the effectiveness of ART. Our study aim to access the extent of latent HIV infections by detecting env V3 DNA and also testing of M184V (meth184val; ATG - GTG substitution at 184th codon) specific mutations in HIV-1 RT gene to monitor the effectiveness of ART. The HIV-1 env V3 DNA sequence was amplified using multiple upstream and downstream primes to show the latent HIV infections, whereas polymerase chain reaction-restriction fragment digestion assay (PCR-RFDA) was used for testing M184V mutation in HIV-1 RT gene. In the study, out of 15 HIV infected patient blood samples, 12 shows amplification of env V3 DNA, confirming the latent HIV infections while 3 were negative for env V3 DNA. HIV-1 RT gene tested for M184V mutation in all 15 samples showed wild-type after analysis using PCR-RFDA. After digestion with CviAII, three bands were observed in wild-type whereas in mutant only two bands. Although the study shows negative for the M184V resistance mutation, screening of various panels of drug resistance mutations should be performed in recently infected HIV-1 patients for planning the effective ART strategy. The data is not enough to compare the overall scenario of Nepal thus warrant urgency for large-scale study with standard genotypic tools.

View Article
December 2016
155 Reads

Variation in Growth Pattern and Morphological Appearance of Primary Monolayer Cultures of Chondrocytes and Neural Cells Isolated from the Chick Embryo at Different Stages

Acta Medica International

Aim: This study was an attempt at investigating the variation in growth and morphology of primary avian chondrocyte and neural cell cultures established under standard laboratory conditions. Methods: For establishing chondrocyte cultures, the tibia were dissected from chick embryos that were 12 (E12), 14 (E14) and 15 (E15) days old. For the neural cell culture initiation, the two lobes of the forebrain were dissected from chick embryos that were 4 (E4), 8 (E8) and 12 (E12) days old. The final characterization of the cells was performed by H&E and CFV staining. Results: In the chondrocyte cultures, two forms of morphology were observed which is reversible in process. The primary variation evaluated in chondrocytes cultures was the effect of the media on the state of the cells. Based on whether the cells were cultured in DMEM or MEM, there was a difference in the transformation of the attached cells from the differentiated to de-differentiated forms. The primary variation examined in neuron cultures was the embryonic age of the tissue and the effect that it had on the proliferation and neurite formation of the cells. E8 embryo neural cells cultures result in well-developed cells with neurite formation along with axonal and dendritic outgrowths with highly complex interconnections between them. Conclusion: Ultimately, this study demonstrated the composition of culture media had an effect on the morphological appearance of the chondrocytes, as well as, confirmed that the culture of primary neurons is best performed using cells from an E8 embryo.

View Article
July 2015

1 Citation

37 Reads

Top co-authors

Yao-Tseng Wen
Yao-Tseng Wen

National Cheng Kung University

3
Rong-Kung Tsai
Rong-Kung Tsai

Buddhist Tzu Chi General Hospital

3
Shailesh Joshi
Shailesh Joshi

Beihang University

1
Alan M Punnoose
Alan M Punnoose

Sri Ramachandra University

1
Asha Palat
Asha Palat

Sri Ramachandra University

1
Anup Bastola
Anup Bastola

Sukraraj Tropical and Infectious Disease Hospital

1
Sundar Khadka
Sundar Khadka

National Public Health Laboratory

1
Sanjeet Pandit
Sanjeet Pandit

National Public Health Laboratory (NPHL)

1
Roshan Pandit
Roshan Pandit

Manmohan Memorial Institute of Health Sciences

1